Single Particle CryoEM of Integral Membrane Proteins

Yifan Cheng
2015 Biophysical Journal  
well as synthetically evolved gain-of-function variants of melittin, which have recently been developed by William Wimley at Tulane University. Using spectroscopic techniques we show that gain-of-function variants are able to leak larger dyes from unilamellar vesicles than melittin. Like melittin gainof-function variants result in virtually complete leakage of the dye from vesicles. However, the concentration of peptide required to achieve leakage is significantly lower for gain-of-function
more » ... ants than for melittin. This suggests that these peptides form larger pores than melittin and that these pores are much more stable, remaining functional over the lifetime of the leakage experiment. Simulations of both melittin and gain-of-function variants reveal a wealth of atomic detail information about transient processes such as peptide absorption, folding, and oligomeric assembly, as well as the equilibrium structural ensemble and stability, which were verified using circular dichroism, fluorescence, and electrochemical impedance spectroscopy. 2513-Plat The Curvature Induction of Surface-Bound Antimicrobial Peptides Piscidin 1 and Piscidin 3 Varies with Lipid Chain Length The initial steps of membrane disruption by antimicrobial peptides (AMPs) involve binding to bacterial membranes in a surface-bound (S) orientation. To evaluate the effects of lipid composition on the S state, molecular dynamics simulations of the AMPs piscidin 1 (p1) and piscidin 3 (p3) were carried out in 4 different bilayers: 3:1 DMPC/DMPG, 3:1 POPC/POPG, 1:1 POPE/POPG, and 4:1 POPC/cholesterol. In all cases, the addition of 1:40 piscidin caused thinning of the bilayer, though thinning was least for DMPC/DMPG. The peptides also insert most deeply into DMPC/DMPG, spanning the region from the bilayer midplane to the head groups, and thereby only mildly disrupting the acyl chains. In contrast, the peptides insert less deeply in the palmitoyl-oleoyl containing membranes, do not reach the midplane, and substantially disrupt the chains; i.e., the neighboring acyl chains bend under the peptide, forming a basket-like conformation. Curvature free energy derivatives calculated from the simulation pressure profiles reveal that the peptides generate positive curvature in membranes with palmitoyl and oleoyl chains but negative curvature in those with myristoyl chains. Curvature inductions predicted with a continuum elastic model follow the same trends, though the effect is weaker and a small negative curvature induction is obtained in POPC/POPG. These results do not directly speak to the relative stability of the inserted (I) states or ease of pore formation, which requires the free energy pathway between the S and I states. Nevertheless, they do highlight the importance of lipid composition and acyl chain packing.
doi:10.1016/j.bpj.2014.11.2729 fatcat:ljg26kqinbcgtbfefidzie5qgq